This invention relates generally to the fields of inorganic and nucleotide chemistry. More specifically, the invention relates to compounds and methods for detecting base pair mismatches in oligonucleotides.
DNA base mismatches arise during the course of genetic recombination and replication as a consequence of enzymatic errors or DNA damage. In the cell, complex systems exist to recognize, remove and repair these mistakes to preserve the integrity of the genetic code. Furthermore, in certain diseases, particularly cancer, these repair systems fail and mismatches persist in a diseased cell""s DNA. The design of molecules and systems site specific recognize mismatches in DNA is an attractive experimental goal both for genetic screening and the design of new chemotherapeutics. Existing strategies include assays using isolated mismatch recognition proteins, hybridization of oligonucleotide-fluorescent probe conjugates, electrophoretic/DNA chip methods, and differential chemical cleavage with reagents assaying for base accessability either in solution or the solid phase. None of these methods are ideal for detection of mismatches in the laboratory, and no strategies exist for using the presence of base mismatches as a way of selective treating disease.
The present invention is based on the discovery of a new class of compounds. These compounds intercalate between bases in a duplex polynucleotide, but only where the bases are not fully complementary, for example, where there is a base mismatch. The compounds are sufficiently hindered that intercalation between bases in fully complementary duplexes does not occur to a significant degree. These mismatch intercalators are useful for detecting DNA and RNA defects, for diagnosing disorders characterized by the presence or increase in DNA and/or RNA defects, and for treating such disorders.
A new class of compounds including Rh(R1)(R2)(R3)3+ and derivatives thereof is here described. The compound intercalates only between nucleotide bases in the presence of a base mismatch, and is useful for detecting single base mismatches. Further, the compound is capable of catalyzing photolytic cleavage of nucleic acids at relatively long wavelengths, and under normal sunlight.
One aspect of the invention is a compound of the formula Rh(R1)(R2)(R3)3+ and derivatives thereof.
Another aspect of the invention is a composition comprising two complementary oligonucleotide strands having a base mismatch, and a compound of the invention.
Another aspect of the invention is a method for determining the presence of a base mismatch in an oligonucleotide, by adding a compound of the invention to a double-stranded oligonucleotide, subjecting the complex to photocleavage conditions, and determining the presence or absence of cleavage products.
Another aspect of the invention is a method for diagnosing a disorder characterized by the presence or increased presence of DNA and/or RNA disorders (for example base pair mismatches), by administering a mismatch intercalator to a cell suspecting of having such a disorder, and determining whether intercalation occurs.
Another aspect of the invention is a method for treating a disorder characterized by the presence or increased presence of DNA and/or RNA disorders (for example base pair mismatches), by administering a compound of the invention to a cell having such disorder, and optionally treating said cell with sufficient light to cause polynucleotide cleavage.
One object of the invention is to provide a method for detecting nucleic acid base mismatches in duplexes.
Another object of the invention is to provide compounds useful for labeling or indicating base mismatches. Another object of the invention is to provide a compound capable of catalyzing cleavage of a duplex having a base mismatch.
Another object of the invention is to provide a method for diagnosing and/or treating a disorder characterized by the presence of base mismatches in nucleic acid duplexes.
Another object of the invention is to provide compounds useful for diagnosing and/or treating disorders characterized by the presence of base mismatches in nucleic acid duplexes.
Another object of the invention is to provide a method for screening agents for their ability to damage nucleic acid duplexes.